Various methods are currently being used in genetic engineering to enable the transfer and expression of genes into the genomes of cells and organisms. Genes have been transferred by incubating cells with DNA, possibly in the presence of chemicals such as polyions or calcium phosphate. Genetic material can also be injected into the nucleus or cytoplasm of cells or zygotes. Other methods include electroporation, liposome mediated gene insertion, asialoglycoprotein gene insertion, particle acceleration and viral transduction.
The use of viruses in the transduction method has been shown to be very efficient when retroviruses are used. Foreign genes are inserted into either a replication defective or replication competent viral vector construct (usually as a plasmid), and are transferred into cells containing all the genes necessary for packaging and replication of the virus. Special cell lines ("helper" or viral packaging cells) have been constructed which enable defective (non-replication competent) viral vectors to be packaged into infectious particles or virions. The vectors themselves do not harbour the necessary genes for replication so that when the vectors infect cells, the vectors replicate using the enzymes in the viral particle to insert themselves into the host genome (chromosomes). The vectors are unable to replicate further because the essential viral genes were (hopefully) left behind in the "helper" cell. This technique has been adopted and approved for the first human gene therapy trials, despite ongoing debate about the safety of such usages.
Transduction by RNA tumor viruses (retroviruses) has special appeal because it can be rapid, efficient, and results in stable integration of a small number of copies (usually 1-2) of genetic information. Unlike transformation, retroviral integration takes place in a precise fashion, usually at an exact position within the viral genome, and insertion can occur at many places within the host cell genome.
Unfortunately, retroviruses are accompanied by adverse pathogenic and oncogenic effects. However, the infectivity of retrovirus vectors can be controlled by using genetically disabled forms in conjunction with helper cells. In order to function effectively as a vector, the portions of necessary genetic information removed from the disabled vector must be provided "in trans" (by another gene, located outside the vector). Many such helper cell lines containing viral functions necessary for packaging, replication, delivery, and reinsertion of replication defective viral vectors have been put into use for the several tumor virus types commonly used. These useful viruses include the disease organisms causing murine leukemia virus (MLV), spleen necrosis virus (SNV), arian leukosis virus (ALV), and reticuloendotheliosis virus (REV). Patents have issued for helper cell lines for MLV and REV (Miller, U.S. Pat. No. 4,861,719; Temin et al., U.S. Pat. No. 4,650,764).
MLV viruses have become the vectors of choice for animal genetic engineering of cells and organisms, because of their compatibility with a wide variety of animal cell types including certain germ cells as well as human cells. MLV was used to insert viral transgenes into the mouse germline, creating a transgenie mouse (Jaenisch et al., Proc. Nat. Acad. Sci. (USA) 73:1260 (1976) and Cell 24:519 (1981)). MLV vector systems have been approved for limited human gene therapy trials despite some of the problems described above.
While retroviruses are becoming important tools for the transfer of genes into cells there are major problems associated with retroviral vectors. One such problem is the ability of disabled viral vector sequences to recombine with either helper viral sequences, or sequences in the cellular genome, yielding infectious viral particles. This problem has been dealt with by removing more viral sequences from vectors, as well as by introducing multiple mutations and rearrangements in helper sequences. Together, these alterations make it more difficult for the vector to revert to replication competency, but may also lower the viral titre or infectivity. However, recombination events are common in retroviruses (Temin, H. M., Genome 31:17-22 (1989)), and hence reversion and recombination persist to be problems.
Another difficulty associated with RNA tumor viruses is that they tend to be oncogenic. Included in this class are the commonly used MLV vectors which cause neoplasms of various sorts by at least two mechanisms: (1) by activating transcription of nearby cellular oncogenes (cancer genes) due at least in part to powerful enhancer-promoter sequences in the viral transcriptional control region, and (2) by recombining with cellular oncogenes, which then become a part of the viral genome, causing rapid oncogenesis that is borne along with the virus.
An additional problem associated with the use of amphotropic (broad host range) vectors such as those being currently proposed for human gene therapy is that the amphotropic vectors might recombine with another retrovirus in the human or animal host, yielding, for example, amphotropic AIDS (AIDS virus able to infect a broad human host range). The possibility theoretically exists for an amphotropic AIDS virus (HIV) arising by recombination or by phenotypic mixing with amphotropic MLV to initiate an epidemic by greatly expanding the range of human cells in which the HIV virus could replicate.
Thus, it is important to assure that non-replication competent MLV vectors used in gene therapy will have a low potential for reversion or pathogenic effects. Ideally, trans-acting viral genes should be completely isolated from vectors, and vectors should be of non-MLV orgin, nonpathogenic, nononcogenic, and bear as little homology to MLVs as possible in order to limit productive recombination which would yield replication competent virus. To be additionally useful for transgenic work, the vectors should be compatible with long-term residence in the genome of the host organism.
The present invention thus relates to the use of natural, mobile cellular genes (retrotransposons) as candidates for gene engineering vectors. Until the present invention, retrotransposons have not been implemented in a practical forum for such uses as animal gene insertion or gene therapy. In contrast, the research is concentrated in other areas. For example, a neomycin phosphotransferase gene as well as the yeast trpl gene in yeast have been amplified after introduction of the genes in a yeast GALl-Ty fusion construct (Boeke et al., Science, 239:280-282, 1988). This involved transposition via Ty element-mediated transposition. Others have developed a means for targeting fusion proteins into yeast Ty retrotransposon particles which can then be harvested and presumably used for the production of antibodies, vaccines, protein purification, diagnostics, and the like. See for example, the Kingsman et al., U.S. Pat. No. 4,918,166. Nonretro transposons (P elements) have also been used for genetic transposition in the fly Drosophila, see for example, the Rubin et al., U.S. Pat. No. 4,670,388. This involves the insertion of DNA material between defined sequences recognized by transposase, inserting the material into a Drosophila germ cell, and causing the transposable element to be affirmatively inserted into the genome of the recipient Drosophila insect so as to become part of the heritable genome of the insect, wherein the element and foreign DNA can function. Mammalian gene transfer and transgenics is discussed in the Wagner et al., U.S. Pat. No. 4,873,191, 1989. This involves introducing exogenous genetic material into a pronucleus of a mammalian zygote by microinjection. The zygote is capable of development into a mammal, and the genetic material includes at least one gene and a control sequence operably associated therewith. Thus, a genetically transformed zygote is obtained. The embryo or zygote is transplanted to a pseudopregnant female and the embryo is allowed to develop to term, wherein the genes are integrated and expressed. This approach is entirely physical and does not involve retrotransposable elements (retrotransposons do not require micro injection). The earliest apparent use of retroviruses to infect the germ line was by Jaenisch et al., Proc. Nat. Acad. Sci. (USA) 73:1260 (1976) and Cell 24:519, (1981). In addition, the Vande Woude et al., U.S. Pat. No. 4,405,712, describes certain vectors derived from Murine leukemia-sarcoma viruses, and the process of transfecting them into cells, selecting phenotype, and then superinfecting cultures in order to obtain replication competent virus with the vectors packaged as pseudotype. These vectors, and the methods described, are quite primitive by todays standards, and do not involve retrotransposon vectors.
Thus, it is clearly advantageous to develop a new, efficient method for the introduction of cellular mobile elements into the genomes of many species. The uses of such method includes, for example, vectors for studies of gene expression in cultured cells, vectors for transgenic organism production and vectors for human gene therapy.